Methods of synthesizing cell-free protein

ABSTRACT

One embodiment of the present invention is a diffusion continuous batch cell-free protein-synthesis method characterized simultaneously by continuously supplying substrate and energy source molecules in the supply phase to the reaction phase by the free diffusion via interface between both phases and by transferring by-products formed in the reaction phase by enhancing the efficiency of the synthesis reaction by prolonging the reaction lifetime by directly contacting a synthesis reaction mixture (reaction phase) containing a biological extract with a substrate- and energy source-supplying solution (supply phase) without using barrier such as semi-permeable membrane or ultrafiltration membrane in a general cell-free protein-synthesis reaction means. Another embodiment of the present invention is a dilution batch cell-free protein synthesis method characterized by enhancing the efficiency of the protein synthesis by prolonging the reaction lifetime by adding a diluting solution to the reaction mixture after pre-incubating the reaction mixture in a cell-free protein-synthesis reaction means using a wheat-embryo extract. Another embodiment of the present invention is a method characterized by enhancing the efficiency of the synthesis reaction simultaneously by re-supplying substrate and energy sources necessary for the protein synthesis (e.g., amino acids, ATP, GTP, creatine phosphate) to the reaction mixture using a gel filtration column and/or semipermeable membrane and by discontinuously removing by-products formed during the reaction after the synthesis reaction stops in the batch cell-free protein synthesis method.

[0001] This application claims priority from Japanese Patent ApplicationNo.2000-259186 which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to a method for synthesizing aprotein using a cell-free system.

BACKGROUND ART

[0003] At present near the completion of the genome project, the centerof the research subject has rapidly been shifting from the genestructural analysis to the gene functional analysis. It is believed thatan intracellular protein does not function singly, but expresses itsfunction cooperatively interacting with various protein factors, nucleicacids, low-molecular species, and cell-membrane components, tobiologically function as the sum of their interactions.

[0004] One of main subjects in the post-genome project is to analyze therelation between structure and function of various protein factorcomplexes. Results obtained from the analyses are expected to providevery important knowledges in wide areas covering basic biologicalstudies including structural biology and biochemistry, elucidation ofthe relation between the gene translation product and the etiology inthe medical field, and the development of medicines.

[0005] As a method for carrying out in vitro the protein-synthesisreaction, so-called “method for cell-free protein synthesis” or the likehas been studied actively (Japan Patent Laid-Open Hei 6-98790, JapanPatent Laid-Open Hei 6-225783, Japan Patent Laid-Open Hei 7-194, JapanPatent Laid-Open Hei 9-291, Japan Patent Laid-Open Hei 7-147992) such asa method in which components containing ribosome or the like that is anintracellular original protein-translating device are extracted from anorganism, and a translation template, amino acids as substrate, energysources, various ions, a buffer, and other effective factors are addedto the extract to synthesize a protein in vitro.

[0006] A cell extract or biological tissue extract for cell-free proteinsynthesis used for the cell-free protein synthesis system is preparedusing Escherichia coli, wheat embryo, and rabbit reticulocyte as a rawmaterial. The cell-free protein-synthesis system maintains theperformance comparable to the living cell with respect to “the peptidesynthesis rate” and “the accuracy of the translation reaction”. Inaddition, the system does not require any complex chemical reaction stepor cell-culturing step. Because of these advantages, practical systemsfor this system have been developed. However, in general, a cell extractobtained from cells of an organism has an extremely unstableprotein-synthesis ability, so that a protein-synthesis efficiency waslow. In addition, the quality of the cell-free extract during storagewas rapidly deteriorated, so that the amount synthesized by a cellprotein-synthesis system was as small as the level that can be detectedby the radio isotope labelling or the like. As a result, this systemcould not be used as a practical means for synthesizing a protein.

[0007] The applicants provided the following methods for solving faultsof the conventional cell-free protein-synthesis system before thisapplication:

[0008] 1) cell extract pharmaceutical preparation for cell-freeprotein-synthesis and method for cell-free protein synthesis(WO00/68412), and

[0009] 2) template molecule that can be widely used and has an efficientfunction and method for cell-free protein-synthesis system using thesame (WO01/27260).

[0010] In addition, a device that carries out the continuous synthesisof a cell-free protein for enhancing the efficiency of the proteinsynthesis. Conventional devices for continuous cell-freeprotein-synthesis apparatus include the ultrafiltration method, thedialysis membrane method, and the column chromatography method using acolumn prepared by fixing a translation template on a resin [Spirin, A.,et al., (1993) Methods in Enzymology, 217, 123-142]. In particular, theultrafiltration method and the dialysis membrane method require onlysimple handling, so that they are widely used. However, these continuousmethods using these membranes have the following problems to solve:

[0011] 1) the material strength of a membrane for use is low,

[0012] 2) the membrane performance can be lowered by the clogging, and

[0013] 3) the operation is complex, so that a skillful technique isnecessary.

[0014] In addition, the continuous cell-free protein-synthesis methodmanually using the ultrafiltration membrane method or the dialysismembrane method can be applied to the protein synthesis from a smallnumber of genes, and it was difficult to efficiently perform the proteinproduction from a large number of genes. Thus, the development of thehigh-throughput full-automatic protein-synthesizing system, forpolyspecimen, which permits efficiently carrying out the proteinproduction from a large number of genes is desired, and the developmentof a new technology in which faults of the conventional continuouscell-free protein-synthesis method are solved is a pressing need.

DISCLOSURE OF THE INVENTION

[0015] According to an aspect of the present invention there is provideda cell-free protein-synthesis method using a diffusion continuous batchsystem that includes a synthesis reaction mixture (reaction phase)containing a biological extract and a supply solution (supply phase)containing substrates and energy sources, the method comprising thesteps of

[0016] 1) bringing the two phases into direct contact with each other tocause a free diffusion via the direct contact interface between the twophases, and continuously supplying substrates and energy sourcemolecules in the supply phase to a translation reaction system in thereaction phase;

[0017] 2) removing by-products formed in the reaction phase; and

[0018] 3) thereby prolonging the lifetime of the synthesis reaction, andenhancing the efficiency of the synthesis reaction.

[0019] The present invention provides the cell-free protein-synthesismethod wherein the biological extract is a wheat-embryo extract.

[0020] The present invention provides the cell-free protein-synthesismethod wherein the biological extract is an Escherichia coli extract.

[0021] The scope of the present invention provides the cell-freeprotein-synthesis method wherein by-products formed in the reactionphase are diluted/removed by transferring to the supply phase.

[0022] The scope of the present invention covers the cell-freeprotein-synthesis method wherein the direct interface formed between thereaction phase and the supply phase is a vertical plane.

[0023] According to another aspect of the present invention there isprovided a cell-free protein-synthesis method comprising the steps ofpre-incubating a cell-free protein-synthesis reaction mixture containinga wheat-embryo extract; and thereafter adding a substrate and energysource-supplying solution to dilute the cell-free protein-synthesisreaction mixture containing a wheat-embryo extract.

[0024] According to a further aspect of the present invention there isprovided a batch cell-free protein-synthesis method comprising the stepsof:

[0025] 1) treating a reaction mixture after the completion of thesynthesis reaction with a gel-filtration column or a semipermeablemembrane;

[0026] 2) re-supplying raw materials and energy sources such as aminoacids, ATP, GTP, and creatine phosphate which are necessary for theprotein synthesis;

[0027] 3) simultaneously with the re-supply, removing by-products formedduring the reaction from the reaction mixture; and

[0028] 4) enhancing the efficiency of the reaction by the removal andthe re-supply.

BRIEF DESCRIPTION OF DRAWINGS

[0029]FIG. 1 illustrates an example of the multilayered method fordiffusion continuous batch cell-free protein synthesis. The partsurrounded by the leader line illustrates a cross section of a well of atitre plate.

[0030]FIG. 2 illustrates the synthesis of green fluorescent protein(GFP) by the diffusion continuous batch cell-free protein synthesismethod using a wheat embryo extract. FIG. 2A illustrates the proteinsynthesis as measured by ¹⁴C-leucine incorporation with Y-axis being anamount of protein synthesized; X-axis an incubation time. The proteinsynthesis was carried out by the conventional batch method (◯-◯), thediffusion continuous batch method (multi-layered method) using areaction vessel having a bore of 7 mm (□-□, large symbol), 5 mm (□-□,middle symbol), or 3 mm (□-□, small symbol), or the dilution batchmethod (▪-▪). The Radioactivity count (Y-axis) showing the amount ofprotein synthesized was shown per the same volume of a wheat embryoextract. FIG. 2B illustrates an autoradiogram of synthetic products.

[0031]FIG. 3 illustrates the synthesis of dihydrofolate reductase (DHFR)by the diffusion continuous batch cell-free protein synthesis methodusing a wheat embryo extract. FIG. 3A illustrates the protein synthesisas measured by ¹⁴C-leucine incorporation by the conventional batch-typemethod (◯-◯) or the diffusion continuous batch method (multi-layeredmethod) using a reactor having a bore of 7 mm (▪-▪). The radioactivitycount (Y-axis) showing the amount of protein synthesized was expressedper the same volume of the embryo extract. FIG. 3B illustrates anSDS-polyacrylamide-gel electropherogram of synthesis products stained byCoomassie Brilliant Blue.

[0032]FIG. 4 illustrates the synthesis of GFP by the diffusioncontinuous batch cell-free protein synthesis method using an Escherichiacoli extract. FIG. 4A illustrates the protein synthesis as measured by¹⁴C-leucine incorporation by the conventional batch method (◯-◯), thediffusion continuous batch method (multi-layered method) using a reactorvessel having a bore of 7 mm (▪-▪), or the dilution batch cell-freeprotein synthesis method (□-□) with Y-axis being the radioactivity countshowing the amount of protein synthesized per the same volume ofEscherichia coli extract. FIG. 4 (B) illustrates an autoradiogram ofsynthesis products.

[0033]FIG. 5 illustrates the synthesis of GFP by the discontinuousgel-filtration batch cell-free protein-synthesis method using a wheatembryo extract (□-□) or without gel filtration (▪-▪). The arrowindicates the time when the reaction mixture was treated with the gelfiltration.

[0034]FIG. 6 illustrates the structure of plasmid pEU1 that can begenerally used.

BEST MODE FOR CARRYING OUT THE INVENTION

[0035] One embodiment of the present invention is a diffusion continuousbatch cell-free protein-synthesis method that consists of a synthesisreaction mixture containing a biological extract (reaction phase) and asubstrate and energy source solution (supply phase), and ischaracterized by:

[0036] 1) continuously supplying substrate and energy source moleculesin the supply phase to the reaction phase by the free diffusion viainterface by directly contacting without barrier such as semipermeablemembrane and ultrafiltration membrane,

[0037] 2) transferring by-products formed in the reaction phase into thesupply phase at the same time as the above supply, and

[0038] 3) prolonging the reaction lifetime by the transfer to enhancethe efficiency of the synthesis reaction.

[0039] The interface between both phases can be formed vertically orhorizontally in the above diffusion continuous batch cell-freeprotein-synthesis method. In order to horizontally form the interface,for example, the reaction phase is added to a reaction vessel to form alower layer, and then the supply phase can be gently overlayed on thereaction phase without disturbing the interface (see FIG. 1). Anyreaction vessel can be used for this purpose as long as the vessel has ashape and a size that give a sufficient diffusion rate for solutes inthe interface. Although tubes and multi-well microtitre plates can beused as such reaction vessels, others can also be used. In addition, theinterface between the two phase can be vertically formed by overlaying asynthesis reaction mixture (reaction phase) on a supply solution (supplyphase), followed by centrifuging a reaction vessel containing these. Thelarger an area of the interface between both phases becomes, the largera mass-exchange rate by the diffusion and a protein-synthesis efficiencybecome. Therefore, the optimal volume ratio of the reaction phase to thesupply phase depends on the area of the interface between both phases.Although any (reaction phase:supply phase) volume ratio can be used, incase the interface is a circle and has a diameter of 7 mm, the (reactionphase:supply phase) volume ratio is preferably between (1:4) and (1:8),more preferably (1:5).

[0040] The synthesis reaction mixture containing the above reactionphase contains a biological extract necessary for the cell-freeprotein-synthesis reaction and a desired mRNA to be a template for theprotein synthesis, and has a composition used for the conventionallyknown batch cell-free protein-synthesis system. For the biologicalextract, well-known biological extracts that have been used for theconventional cell-free protein-synthesis method can be used such aswheat embryo extract, Escherichia coli extract, and rabbit reticulocyteextract. These extracts can be prepared according to well-known methods.A wheat embryo extract is preferably prepared according to the methoddescribed in Madin K., et al., Proc. Natl. Acad. Sci. USA (2000), 97,559-564 (WO00/68412). The synthesis reaction mixture contains abiological extract, for example, at 48%(v/v) per total volume(200A_(260nm) units/ml) in case a wheat embryo extract is used as thebiological extract, and has the following composition (finalconcentration): 1,000 units/ml ribonuclease inhibitor (RNAsin), 30 mMHEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate,2.85 mM dithiothreitol, 0.5 mg/ml creatine kinase, 1.2 mM adenosinetriphosphate (ATP), 0.25 mM guanosine triphosphate (GTP), 16 mM creatinephosphate, 0.380 mM spermidine, 0.3 mM each of twenty L-type aminoacids, 0.05% NP-40, and 600 μg/ml mRNA. The composition of the synthesisreaction mixture is not limited to the above composition. Anycomposition can be used as long as the cell-free protein-synthesisreaction efficiently progresses. For example, instead of the above mRNA,mRNA is synthesized by a transcription reaction mixture containing aplasmid (encoding the target gene), RNA polymerase, nucleotides, and soon, and the composition of the transcription reaction mixture is changedby the gel filtration method and/or the dialysis method to one suitablefor the translation, and the obtained solution can be used as thesynthesis reaction mixture (see Example 1 below).

[0041] The above composition of the synthesis reaction mixture can beproperly changed depending on the kind of the biological extract to use.When Escherichia coli is used as the biological extract, aprotein-synthesis reaction mixture can be prepared by the methoddescribed by Pratt, J. M. [Transcription and Translation (1984),179-209, Hames, B. D. & Higgins, S. J., eds. IRL Press, Oxford] using anE. coli extract prepared according to the literature. For example, atranscription reaction mixture is prepared that contains an E. coliextract at 50%(v/v), and has the following composition (finalconcentration): 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mMammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol, 0.3U/ml pyruvate kinase, 0.17 mg/ml E. coli tRNA mixed solution, 34 mg/mlL-5-formyl-5,6,7,8-tetrahydrofolic acid, 6.7 μg/ml plasmid (encoding thetarget gene), 33 μg/ml T7 RNA polymerase, 1.2 mM ATP, 0.85 mM GTP, 0.85mM UTP, 0.85 mM CTP, 56 mM phosphoenolpyruvate, and 0.2 mM each oftwenty L-type amino acids to synthesize a mRNA, and then the compositionof the obtained transcription reaction mixture is changed by the gelfiltration method and/or the dialysis method to a mixture having acomposition suitable for the translation reaction, and the obtainedsolution can be used as the synthesis reaction mixture (see Example 1below).

[0042] In the case of a cell-free protein-synthesis system using an E.coli extract, an mRNA is synthesized with a transcription reactionmixture as described above, and a supply solution is overlayed onto thetranscription reaction mixture, and then a protein synthesis reactioncan be carried out under a stationary condition at a temperaturesuitable for the translation reaction. The composition of the synthesisreaction mixture is not limited to the above composition, but anycomposition can be used as long as the cell-free protein-synthesisreaction efficiently progresses. For example, a synthesis reactionmixture having a composition suitable for the translation reaction canbe prepared by properly adding an mRNA encoding the target gene preparedaccording to a well-known method (Gurevich, V.V., (1996) Methods inEnzymology, 275, 383-397) instead of plasmid (encoding the target gene),T7 RNA polymerase, UTP, and CTP in the above transcription reactionmixture.

[0043] In addition, the protein synthesis reaction can be furtherstabilized by adding a sugar alcohol such as inositol, xylitol and/orficoll to the above synthesis reaction mixture to elevate the viscosityor density of the synthesis reaction mixture to control the mixing ratebetween the reaction phase and the supply phase.

[0044] Moreover, the supply solution forming the above supply phasecontains substrates and energy sources (e.g., amino acids, ATP, GTP,creatine phosphate), other ions necessary for the protein synthesis, anda buffer. For example, in case the above protein synthesis reactionmixture containing a wheat embryo extract is used as the reaction phase,a supply solution containing 30 mM HEPES-KOH (pH 7.6), 95 mM potassiumacetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 1.2 mM ATP,0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine and 0.3 mMeach of twenty L-type amino acids can be used. In case the aboveprotein-synthesis reaction mixture containing an Escherichia coliextract is used as the reaction phase, for example, a supply solutioncontaining 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mMammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol, 34mg/ml L-5-formyl-5,6,7,8-tetrahydrofolic acid, 1.2 mMATP, 0.85 mM GTPand UTP and CTP, 56 mM phosphoenolpyruvate, and 0.2 mM each of twentyL-type amino acids, can be used.

[0045] The protein synthesis reaction is carried out under a stationarycondition at an optimal temperature usually used for various cell-freeprotein-synthesis reactions. The temperature is from 20° C. to 30° C.,preferably 26° C. in case a wheat embryo extract is used as thebiological extract, while the temperature is from 30° C. to 37° C.,preferably 30° C. in case an Escherichia coli extract is used as thebiological extract.

[0046] In one embodiment of the present invention, in a cell-freeprotein-synthesis method using a wheat-embryo extract, a synthesisreaction mixture is diluted by adding a diluting solution after thereaction mixture is pre-incubated. Thus, the synthesis-reaction lifetimecan be prolonged to enhance the efficiency of the protein synthesis.

[0047] In a dilution batch cell-free protein-synthesis method, aconventional batch cell-free protein-synthesis reaction mixture, forexample, using a synthesis reaction mixture having the above compositionis used, after pre-incubation is carried out for 15 min to 30 min, theprotein synthesis is carried out. Then, a wheat-embryo extract containedin the reaction mixture is diluted to 7%-12% or so by adding “a solutionhaving the same composition as the supply solution in the abovediffusion continuous protein-synthesis method” containing substrates andenergy sources (e.g., amino acids, ATP, GTP, creatine phosphate), otherions necessary for the protein synthesis, and a buffer. Then thereaction is carried out under the conditions. The optimal temperaturefor the protein synthesis reaction is from 20° C. to 30° C., preferably26° C. in case a wheat-embryo extract is used. It is known that thestability of enzymes and translation protein factors generally decreasesat low concentrations. Therefore, the efficiency of the synthesisreaction can be further enhanced by beforehand adding well-knownstabilizer(s) such as inositol, xylitol and ficoll to the synthesisreaction mixture.

[0048] Although the above dilution batch protein synthesis method usinga wheat-embryo extract was very effective, the effect could not beconfirmed with a system using an Escherichia coli extract probably dueto properties of the wheat-embryo extract.

[0049] Moreover, pre-incubation is an extremely important step in thismethod, and omitting this step reduces the efficiency of the proteinsynthesis reaction. Therefore, it is considered that a stabletranslation-initiation complex is formed during the pre-incubation.However, the molecular mechanism concerning this peculiar phenomenon isa future subject.

[0050] In addition, one embodiment of the present invention is a batchcell-free protein-synthesis method characterized by:

[0051] 1) using “a gel-filtration column or semipermeable membrane” forthe reaction mixture after the synthesis reaction stops,

[0052] 2) re-supplying raw materials necessary for the protein synthesissuch as substrates and energy sources necessary for the proteinsynthesis (e.g., amino acids, ATP, GTP, creatine phosphate)

[0053] 3) at the same time as (2), removing by-products formed duringthe reaction from the reaction mixture, and

[0054] 4) enhancing the efficiency of the synthesis reaction.

[0055] This method is a batch method comprising discontinuous steps ofthe protein-synthesis reaction, the supply of substrates and energysources to the reaction mixture, and the removal of by-products. Thismethod is fundamentally different from the continuous cell-freeprotein-synthesis method according to Spirin et al.

[0056] In this discontinuous batch cell-free protein-synthesismethod, 1) a conventional batch cell-free protein-synthesis reaction isinitiated using a reaction vessel such as test tube, 2) theprotein-synthesis reaction is completely stopped by chilling thereaction mixture down to 0° C. to 4° C. after the synthesis reactionstopped, and 3) the reaction mixture after the reaction stop is treatedwith the column chromatography using gel filtration particles (e.g.,Sephadex G-25) for separating low-molecular compounds beforehandequilibrated with a solution containing substrates and energy sourcessuch as amino acids, ATP, GTP, and creatine phosphate. For theequilibration, a solution having the same composition as the supplysolution in the above diffusion continuous batch protein-synthesismethod can be used.

[0057] By the above gel-filtration procedure, by-products are trapped inSephadex particles, and a cell-free protein-synthesis reaction mixtureexchanged with fresh amino acids, ATP, GTP, and creatine phosphate isregenerated in a void fraction. Incubating the regenerated solutionagain permits initiating the translation reaction, and theprotein-synthesis reaction progresses for several hours. In case thesynthesis reaction is stopped again, the above gel-filtration procedureis repeated. By repeating this procedure, the synthesis reaction that isstopped in a short time by a usual batch method can be continued for along time to enhance the protein-synthesis yield.

[0058] Moreover, even if the dialysis method is used instead of theabove gel-filtration method for “the re-supply of substrates and energysources” and “the removal of by-products” in the above discontinuousbatch cell-free protein-synthesis method, a similar effect or higher isobtained.

[0059] As described above, the batch method according to the presentinvention is:

[0060] 1) a diffusion continuous batch method using a reaction phaseconsisting of a synthesis reaction mixture containing a cell-freeextract and a supply phase consisting of a substrate- andenergy-source-supplying solution containing amino acids, ATP, GTP, andcreatine phosphate, wherein a) the reaction phase and the supply phaseare directly contacted each other to continuously supply substrates andenergy sources into the reaction phase by the free diffusion via theinterface, and b) at the same time as the above supply, by-productsformed in the reaction phase are transferred into the supply phase,

[0061] 2) a dilution batch method by reducing the concentration of thecell extract contained in the synthesis reaction mixture in a cell-freeprotein-synthesis system using a wheat embryo extract, and

[0062] 3) a discontinuous batch method by using the gel-filtrationmethod or the dialysis method after the protein-synthesis reaction isstopped, wherein a) substrates and energy sources such as amino acids,ATP, GTP, and creatine phosphate necessary for the protein synthesis arere-supplied to the synthesis reaction mixture, and b) at the same timeas the above re-supply, by-products formed during the reaction arediscontinuously removed.

[0063] The above batch method according to the present invention isextremely effective as a cell-free protein-synthesis method differentfrom the conventional continuous batch cell-free protein-synthesismethod.

[0064] These methods can be carried out singly or in a combination. Forexample, the combination of the diffusion continuous batch method andthe discontinuous batch method or the combination of the dilution batchmethod and the discontinuous batch method can be carried out forenhancing the efficiency of the protein synthesis. Moreover, theconcentration of a cell or tissue extract to initially add can beelevated to carry out the combination of the above three methods.

[0065] In addition, the present invention permitted prolonging thelifetime of the cell-free protein-synthesis reaction, hence remarkablyenhancing the efficiency of the protein synthesis compared with theconventional batch method, and establishing the cell-freeprotein-synthesis method having a performance comparable or more to thecontinuous cell-free protein-synthesis method, using a semipermeablemembrane, established by Spirin et al. [Spirin, A., et al., (1993)Methods in Enzymology, 217, 123-142].

EXAMPLES

[0066] Although the present invention is described in more detail citingexamples below, the present invention is not limited to the examplesbelow.

Example 1

[0067] As an example of a diffusion continuous batch cell-freeprotein-synthesis method, the protein synthesis was carried out using awheat-embryo extract by the multi-layered diffusion continuous batchmethod as illustrated in FIG. 1.

[0068] The wheat embryo extract was prepared according to Madin K. etal. [Proc. Natl. Acad. Sci. USA (2000), 97, 559-564; WO00/68412].

[0069] In addition, in order to synthesize an mRNA to be a translationtemplate in the wheat-embryo cell-free protein-synthesis reaction,general plasmid pEU1 constructed by Endo (FIG. 6) (WO01/27260) was used.As a gene encoding the target protein, jellyfishgreen-fluorescent-protein (GFP) gene (gfp gene) was used, and wasinserted into the above plasmid according to the conventional method.The obtained plasmid was cut with HindIII to give a linear form, whichwas used as a translation template to synthesize an mRNA according tothe conventional method. The synthesized mRNA does not have CAP at5′-end, has AMV-Ω sequence at 5′-end as non-translation sequence, andhas 500 bases derived from the plasmid at 3′-end. The above AMV-Ωsequence is a base sequence obtained by serially linking 5′-end leaderstructure of alfalfa mosaic virus mRNA (AMV-mRNA) to 5′-end Ω sequenceof tobacco mosaic virus mRNA (TMV-mRNA) (WO01/27260). The addition ofthese non-translation sequence enhances the stability of the RNA, sothat the use of this mRNA enhances the efficiency of the cell-freeprotein synthesis. Moreover, the use of an mRNA having CAP at 5′-endalso gave the result similar to one below.

[0070] Then, a protein-synthesis reaction mixture that contains 48%(v/v)wheat-embryo extract (concentration of 200A_(260nm) units/ml), 1,000units/ml ribonuclease inhibitor (RNAsin) (Takara Bio Inc.), 30 mMHEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate,2.85 mM dithiothreitol, 0.5 mg/ml creatine kinase, 1.2 mM adenosinetriphosphate (ATP), 0.25 mM guanosine triphosphate (GTP), 16 mM creatinephosphate, 0.380 mM spermidine, 0.3 mM each of twenty L-type aminoacids, 0.05% NP-40, and 600 μg/ml mRNA, was prepared. In order to assaythe amount of protein synthesized, 4 μCi of ¹⁴C-leucine (300 mCi/mmol)was added per 1 ml of the above protein synthesis reaction mixture[Proc. Natl. Acad. Sci. USA (2000), 97, 559-564].

[0071] This protein-synthesis reaction mixture was added to reactionvessels having a bore of 7 mm (microtitre plate), 5 mm (1.5-ml testtube), and 3 mm (0.2-ml test tube), and 5 times volumes of supplysolution [30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mMmagnesium acetate, 2.85 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16mM creatine phosphate, 0.380 mM spermidine and 0.3 mM each of twentyL-type amino acids] were overlayed on the reaction mixture gentlywithout disturbing the interface, and the resultant preparation wasincubated under a stationary condition at 26° C. for 3, 6, 9, or 17 h tocarry out the protein synthesis reaction. The amount of proteinssynthesized was assayed using the incorporation of a radio isotope intoa trichloroacetic acid-insoluble fraction as an index according to theconventional method, and the synthesized proteins were visualized by theautoradiography [Endo, Y. et al., (1992) J. Biotechnol., 25, 221-230][Proc. Natl. Acad. Sci. USA(2000), 97, 559-564]. Results are shown inFIG. 2A and FIG. 2B.

[0072] As a control, the conventional batch cell-free protein-synthesismethod was carried out. In this method, the same mRNA, wheat-embryoextract, and protein-synthesis reaction mixture containing these asthose used in the above diffusion continuous batch cell-freeprotein-synthesis method were used except that the supply solution wasnot added.

[0073] As FIG. 2A illustrates, the protein-synthesis reaction stopped 1h after the initiation of the reaction in the conventional batch method(◯-◯). This result is in complete agreement with the results of Endo, Y.et al., (1992) J. Biotechnol., 25, 221-230 and Proc. Natl. Acad. Sci.USA (2000) 97, 559-564.

[0074] On the other hand, in the multi-layered method using a reactionvessel having a bore of 7 mm (area of the interface is 0.385 cm²) (□-□,large symbol), the synthesis reaction progressed even 17 h after theinitiation of the reaction, the amount synthesized reached 9 times ormore that of the conventional batch method. In addition, the influenceof the area of the interface between the reaction phase and the supplyphase on the synthesis reaction was investigated using reaction vesselshaving different sizes, the synthesis efficiency in 9 h after theinitiation of the reaction was showed that a reaction vessel having abore of 5 mm (area of the interface was 0.196 cm²) (□-□, middle symbol)was 91% as compared with that of 7 mm, a reaction vessel having a boreof 3 mm (area of the interface was 0.071 cm²) (□-□, small symbol) was75% as compared with that of 7 mm.

[0075] The autoradiogram shown in FIG. 2B completely supported theexperimental result obtain in the investigation of the amount of proteinsynthesized as measured by ¹⁴C-leucine incorporation shown in FIG. 2Awith respect to both “the synthesis reaction lifetime transient ofproteins” and “molecular weight of synthesis products and amountthereof” in the conventional batch method and the diffusion continuousbatch method. The diffusion continuous batch method is displayed as ‘theoverlay method’ in FIG. 2B. Moreover, the protein synthesis results bythe diffusion continuous batch method are shown with respect only toresults using reaction vessels having a bore of 7 mm.

[0076] Similar results were obtained also in case of synthesizing anmRNA with a transcription reaction solution according to thetranscription-translation all-in-one protein-synthesis method shown inReference Example 1 below, following by changing the composition of thetranscription reaction mixture to a solution having a compositionsuitable for the translation reaction by the gel-filtration methodand/or the dialysis method, the obtained solution was used as asynthesis reaction mixture according to a manner similar to thatdescribed above in protein synthesis.

[0077] The above results showed that 1) the diffusion continuous batchprotein synthesis method using a wheat embryo extract gives a remarkablyhigh synthesis efficiency compared with the conventional bath method,that 2) the larger the area of the interface between the reaction phaseand the supply phase is, the higher the synthesis efficiency is, andthat 3) the increase in the synthesis yield by the method is due to theprolongment of the synthesis-reaction lifetime.

Example 2

[0078] As one example of the dilution batch cell-free protein-synthesismethod, the protein-synthesis reaction was carried out by carrying out apre-incubation using a protein-synthesis reaction mixture containing thewheat embryo extract prepared in Example 1 and an mRNA encoding GFP bythe conventional batch method at 26° C. for 15 min, adding five timesvolumes of the diluting solution to the resultant mixture, followed byfurther incubating the diluted mixture at 26° C. for 3, 6, or 9 h,wherein the diluting solution had the same composition as the supplysolution prepared in Example 1, wherein the amount of proteinsynthesized was assayed in a manner similar to that of Example 1.Results are shown in FIG. 2A (▪-▪) and FIG. 2B.

[0079] As FIG. 2A shows, compared with the conventional batch method inwhich the synthesis reaction stops within 1 h (◯-◯), in case thecell-free protein synthesis was carried out by the dilution batchmethod, the synthesis reaction linearly progressed for 6 h after theinitiation of the reaction (▪-▪).

[0080] The autoradiogram shown in FIG. 2B completely supported theexperimental results obtained in the investigation of the amount of theprotein synthesized as measured by the ¹⁴C-leucine incorporation shownin FIG. 2A.

[0081] Although this dilution batch cell-free protein-synthesis methodgives a lower synthesis yield than that of the diffusion continuousbatch method shown in Example 1, the amount of the protein synthesizedby the dilution batch cell-free protein synthesis method was about threetimes that of the conventional batch method, and the dilution batchcell-free protein-synthesis method showed a significantly high synthesisefficiency.

[0082] Similar results were obtained also in case of synthesizing anmRNA with a transcription reaction solution according to thetranscription-translation all-in-one protein-synthesis method shown inReference Example 1 below, following by changing the composition of thetranscription reaction mixture to a solution having a compositionsuitable for the translation reaction by the gel-filtration methodand/or the dialysis method, the obtained solution was used as asynthesis reaction mixture according to a manner similar to thatdescribed above in protein synthesis.

[0083] Moreover, in case the pre-incubation reaction procedure wasomitted in the above dilution batch cell-free protein-synthesis method,the above remarkable prolongment of the synthesis-reaction lifetime wasnot observed. In addition, with respect to the cell-freeprotein-synthesis system using an Escherichia coli extract, the effectof the dilution batch method was not recognized.

[0084] As described above, it was demonstrated that the dilution batchcell-free protein-synthesis method is also an effectiveprotein-synthesis means with respect to the cell-free protein-synthesissystem using a wheat-embryo extract.

Example 3

[0085] It was demonstrated that a dihydrofolate reductase (DHFR) derivedfrom Escherichia coli can be synthesized in addition to GFP synthesizedin Example 1 by the diffusion continuous batch cell-freeprotein-synthesis method, and it was confirmed that this method iseffective for the synthesis of general protein molecule species.

[0086] The protein synthesis was carried out in a manner similar toExample 1 using a protein-synthesis solution having a compositionsimilar to that of Example 1 except that the mRNA was an mRNA encodingDHFR, giving the results shown in FIG. 3. The amounts of proteinssynthesized were assayed using the incorporation of the radio isotopeinto a trichloroacetic acid-insoluble fraction as an index according tothe conventional method, and proteins synthesized were identified by theSDS-polyacrylamide-gel electrophoresis and the staining with CoomassieBrilliant Blue (CBB) [Endo, Y. et al., (1992) J. Biotechnol., 25,221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564].

[0087] As shown in FIG. 3A, also in the DHFR synthesis, the synthesisreaction by the diffusion continuous batch cell-free protein-synthesismethod (▪-▪) significantly continued longer than that of theconventional batch method (◯-◯). Results of the analysis of the lifetimeof the synthesis reaction and the amounts of synthesis products by theSDS-polyacrylamide-gel electrophoresis [arrows in FIG. 3B show bands ofDHFR stained with Coomasie Brilliant Blue] completely supported theresults shown in FIG. 3A. The measurement of the band staining intensityof synthesized DHFR demonstrated that the diffusion continuous batchcell-free protein-synthesis method permitted synthesizing 0.9 mg DHFRper 1 ml of reaction volume for 8 h.

Example 4

[0088] It was demonstrated by using an Escherichia coli extract that thediffusion continuous batch cell-free protein-synthesis method isgenerally effective even for a cell-free protein-synthesis system usinga cell extract prepared from any biological species.

[0089] The Eseherichia coli extract was prepared according to Pratt, J.M., Transcription and Translation (1984), 179-209, Hames, B. D. &Higgins, S. J., eds, IRL Press, Oxford. In this example, a mRNA wasfirst synthesized by the transcription-translation all-in-one cell-freeprotein-synthesis system (see Reference Example 1), and then thecomposition of the transcription reaction mixture containing the mRNAwas changed to a composition suitable for the translation reaction bythe gel filtration method, and the resultant mixture was added to areaction vessel, and a supply solution was overlayed onto the mixture ina manner similar to Example 1, and the obtained preparation wasincubated under a stationary condition at 30° C. to carry out theprotein synthesis.

[0090] An mRNA was synthesized by preparing an Escherichia colicell-free protein-synthesis reaction mixture that contains an E. coliextract at 50%(v/v) and has the following composition (finalconcentration): 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mMammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol, 0.3U/ml pyruvate kinase, 0.17 mg/ml E. coli tRNA mixed solution, 34 mg/mlL-5-formyl-5,6,7,8-tetrahydrofolic acid, 6.7 μg/ml plasmid (encoding GFPgene), 33 μg/ml T7 RNA polymerase, 1.2 mM ATP, 0.85 mM GTP and UTP andCTP, 56 mM phosphoenolpyruvate, and 0.3 mM each of twenty L-type aminoacids, followed by incubating the resultant mixture at 30° C. for 90min. Then, the composition of the above reaction mixture was changed toa composition suitable for the translation reaction by the gelfiltration method, and then 25 μl of the obtained mixture wastransferred to a reaction vessel (microtitre plate having a bore of 7mm), and the supply solution having the following composition was gentlyoverlaid, and the obtained preparation was incubated at 30° C. to carryout the protein synthesis. In case the protein synthesis was measuredusing the incorporation of an amino acid as index, 4 μCi of ¹⁴C-leucine(300 mCi/mmol) was added to 1 ml of the above reaction mixture.

[0091] The plasmid to be the transcription template of an mRNA wasprepared by replacing RAS gene of pK7-RAS [Kigawa, T., et al., (1995) J.Biomol. NMR, 6, 129-134] having T7-phage promoter sequence with jellyfish GFP gene.

[0092] The composition of the supply solution used for the Escherichiacoli cell-free protein synthesis system was as follows (finalconcentration): 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mMammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol, 34mg/ml L-5-formyl-5,6,7,8-tetrahydrofolic acid, 1.2 mM ATP, 0.85 mM GTPand UTP and CTP, 56 mM phosphoenolpyruvate, and 0.2 mM each of twentyL-type amino acids. In case the protein synthesis was measured using theincorporation of an amino acid as index, 4 μCi of ¹⁴C-leucine (300mCi/mmol) was added to 1 ml of the above reaction mixture.

[0093]FIG. 4A illustrates results of the protein synthesis as measuredby the incorporation of ¹⁴C-leucine. GFP synthesis reaction by theconventional batch method (◯-◯) completely stopped 3 h after theinitiation of the reaction, while that of the diffusion continuous batchcell-free protein-synthesis method (▪-▪) lasted for 17 h after theinitiation of the reaction. The amount of synthesized protein ascalculated from the amount of incorporated amino acid in the diffusioncontinuous cell-free protein synthesis method reached 4 times or morethat of the batch method. Results of the analysis of the time transientof the synthesis reaction and molecular weights and synthesis amounts ofsynthesis products by the autoradiogram shown in FIG. 4B completelysupported results of FIG. 4A. On the other hand, in the controlexperiment in which both phases were mixed by a vortex mixer just afterthe above supply solution (supply phase) was overlayed onto the aboveprotein-synthesis reaction mixture (reaction phase), the proteinsynthesis was significantly reduced compared with the conventional batchmethod.

[0094] This fact is in complete agreement with the result thatincreasing the concentration of a cell-free extract in the reactionmixture with respect to the Escherichia coli cell-free protein synthesissystem is important for enhancing the efficiency [Kim, D. M., (1996)Eur. J. Biochem. 239, 881-886]. This result clearly shows that theprolongment of the lifetime of the protein-synthesis reaction observedin the diffusion continuous batch method is not due to, for example, thereduction of the reaction rate by the reduction of the concentration ofa component necessary for the protein synthesis in the reaction mixture,such as, ribosome, but due to the intrinsic property of the diffusioncontinuous batch cell-free protein synthesis method.

[0095] In addition, results similar to those described above wereobtained also by synthesizing an mRNA by the transcription-translationall-in-one cell-free protein synthesis system (see Reference Example 1)using an Escherichia coli extract, overlaying the supply solution ontothe reaction mixture in a manner similar to Example 1, followed bycarrying out the protein-synthesis reaction under a stationary conditionat 30° C.

Example 5

[0096] An example of the cell-free protein-synthesis method using awheat embryo by the discontinuous batch method using the gel filtrationmethod is described below. The same protein-synthesis solution asprepared in Example 1 was added to normal small test tubes or a 96-welltitre plate, and the obtained preparation was incubated under astationary condition at 26° C. by a usual method. Under this reactionconditions, the protein synthesis stops within several hours. Forexample, in case a reaction mixture containing a wheat embryo extract at48%(v/v) is used, the synthesis reaction completely stops within 1 hafter the initiation of the reaction. This can be confirmed by themeasurement of amino acid incorporation into proteins and/or thepolyribosome analysis by the sucrose density gradient centrifugationmethod [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564]. The reactionmixture after the above synthesis reaction stopped was treated with thegel filtration using a Sephadex G-25 column beforehand equilibrated witha supply solution containing substrates and energy sources (e.g., aminoacids, ATP, GTP), other ions necessary for the protein synthesisreaction, and a buffer, and the protein synthesis was carried out againat 26° C., wherein the supply solution had a composition similar to thatof Example 1.

[0097] As the result of ¹⁴C-leucine incorporation in FIG. 5 shows, thesynthesis reaction by the conventional batch method completely stoppedwithin 1 h after the initiation of the reaction (▪-▪). However, the gelfiltration as described above 3 h after the initiation of the reaction(arrow in FIG. 5) and the subsequent incubation permitted the restart ofthe protein synthesis reaction (□-□). In addition, the velocity gradientof ¹⁴C-leucine incorporation was almost equal to that in the early stageof the reaction. Therefore, it was found that the efficiency of theprotein synthesis after the gel filtration was at a level comparable tothat in the early stage of the reaction.

[0098] Similar results were obtained also in case of synthesizing anmRNA with a transcription reaction solution according to thetranscription-translation all-in-one protein-synthesis method shown inReference Example 1 below, following by changing the composition of thetranscription reaction mixture to a solution having a compositionsuitable for the translation reaction by the gel-filtration methodand/or the dialysis method, the obtained solution was used as asynthesis reaction mixture according to a manner similar to thatdescribed above in protein synthesis.

[0099] Since the above cell-free protein-synthesis system using awheat-embryo extract is extremely stable [Endo, Y et al., (1992) J.Biotech., 25, 221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564],it is possible to keep the reaction time for a long time by repeatingthe gel filtration procedure. Therefore, the above discontinuous batchcell-free protein-synthesis method is useful as an efficient proteinsynthesis method, for example, in a cell-free protein-synthesis systemusing a wheat-embryo extract.

Reference Example 1

[0100] The transcription-translation all-in-one protein-synthesis methodpermits synthesizing an mRNA with the transcription reaction mixture,then changing the composition of the transcription reaction-mixture toone suitable for the translation reaction by the gel filtration methodand/or the dialysis method, and using the obtained mixture as thesynthesis reaction mixture.

[0101] First, a transcription reaction mixture consisting of a templateDNA, four kinds of substrate ribonucleoside-5′-triphosphate, and ifnecessary, CAP molecule, RNA polymerase, spermidine, magnesium ion, andan appropriate buffer [80 mM HEPES-KOH (pH 7.6), 16 mM magnesiumacetate, 2 mM spermidine, 10 mM dithiothreitol, 2.5 mM ATP, 2.5 mM GTP,2.5 mM CTP, 2.5 mM UTP, 1U/μl ribonuclease inhibitor, 3U/μl SP6 RNApolymerase (Takara Bio., Inc)] was added to a gel filter-installed spincolumn as the reaction vessel. An mRNA is continuously synthesized bythe dialysis using an outer solution, for the dialysis, which has thesame composition as the above transcription reaction mixture excepttemplate DNA, RNA polymerase, and ribonuclease inhibitor.

[0102] After the synthesis of the mRNA, the spin column is centrifugedat a low speed, and the composition of the above transcription reactionmixture is changed to one suitable for the translation reaction by thegel filtration procedure using the protein synthesis solution (notcontaining mRNA) as shown in Example 1.

INDUSTRIAL APPLICABILITY

[0103] The above cell-free protein-synthesis method according to thepresent invention does not require complicated techniques such as theultrafiltration method and dialysis-membrane method using asemipermeable membrane and the column chromatography using a resin towhich a translation template was fixed [Spirin, A., et al., (1993)Methods in Enzymology, 217, 123-142]. Therefore, it was shown that theabove cell-free protein synthesis method according to the presentinvention permits efficiently synthesizing a protein in a cell-freesystem using a tissue/cell extract by any means by introducing one ofthree techniques for making the synthesis reaction efficient into theconventional batch method.

[0104] The above cell-free protein synthesis method according to thepresent invention does not have faults such as weakness of the materialstrength of the membrane, decrease in the membrane function due to theclogging, and complexity of the operation, which are observed in theconventional continuous cell-free protein synthesis method using amembrane. Thus, the above cell-free protein-synthesis method accordingto the present invention permits carrying out the protein synthesis at aconsiderably high efficiency compared with the conventional method.Therefore, the above technique according to the present invention wouldbe a fundamental element technology, for the automation of theproduction of a gene product (protein), which is the base for thefunctional analysis and the structural analysis of a great number ofgenes that would be provided till the future completion of the genomeproject, in particular be an essential element technology for theautomation of the cell-free protein-synthesis system such as developmentof full-automatic cell-free protein-synthesis robot for polyspecimen.

What is claimed is:
 1. A cell-free protein-synthesis method using adiffusion continuous batch system that includes a synthesis reactionmixture (reaction phase) containing a biological extract and a supplysolution (supply phase) containing substrates and energy sources, themethod comprising the steps of 1) bringing the two phases into directcontact with each other to cause a free diffusion via the direct contactinterface between the two phases, and continuously supplying substratesand energy source molecules in the supply phase to a translationreaction system in the reaction phase; 2) removing by-products formed inthe reaction phase; and 3) thereby prolonging the lifetime of thesynthesis reaction, and enhancing the efficiency of the synthesisreaction.
 2. A cell-free protein-synthesis method according to claim 1,wherein the biological extract is a wheat-embryo extract.
 3. A cell-freeprotein-synthesis method according to claim 1, wherein the biologicalextract is an Escherichia coli extract.
 4. A cell-free protein-synthesismethod according to claim 1, wherein by-products formed in the reactionphase are diluted/removed by transferring to the supply phase.
 5. Acell-free protein-synthesis method according to claim 1, wherein thedirect interface formed between the reaction phase and the supply phaseis a vertical plane.
 6. A cell-free protein-synthesis method comprisingthe steps of: pre-incubating a cell-free protein-synthesis reactionmixture containing a wheat-embryo extract; and thereafter adding asubstrate and energy source-supplying solution to dilute the cell-freeprotein-synthesis reaction mixture containing a wheat-embryo extract. 7.A batch cell-free protein-synthesis method comprising the steps of 1)treating a reaction mixture after the completion of the synthesisreaction with a gel-filtration column or a semipermeable membrane; 2)re-supplying raw materials and energy sources such as amino acids, ATP,GTP, and creatine phosphate which are necessary for the proteinsynthesis; 3) simultaneously with the re-supply, removing by-productsformed during the reaction from the reaction mixture; and 4) enhancingthe efficiency of the reaction by the removal and the re-supply.